Molecular markers and methods for early sex determination in date palms

Abstract

The present invention relates to molecular markers and to the use thereof for distinguishing male date palm plants from female date palm plants. The invention also relates to a method for identifying the sex of date palm plants using these molecular markers, and to a kit for carrying out the method.

1 . A method for identifying the sex of a date palm by detection, using a microsatellite marker, of an SSR polymorphism in the date palm genome, the method comprising steps of:
 amplifying a portion of genomic DNA of the date palm tested using a pair of primers specific of the microsatellite marker in order to obtain amplicons, and   analyzing the amplicons obtained in order to determine the sex of the date palm tested,   
       wherein the microsatellite marker is selected from the group consisting of:
 the P80 microsatellite marker which, in the 5′→3′ direction, has a (ga) motif and is flanked, in 5′, by the nucleotide sequence occupying positions 73550 to 74549 in the PDK — 30s6550963 scaffold (GenBank accession number: GL739764.1), and in 3′, by the nucleotide sequence occupying positions 74566 to 75565 in the PDK — 30s6550963 scaffold; 
 the P50 microsatellite marker which, in the 5′→3′ direction, has an (ag) motif and is flanked, in 5′, by the nucleotide sequence occupying positions 4871 to 5870 in the PDK — 30s1202771 scaffold (GenBank accession number: GL744456.1), and in 3′, by the nucleotide sequence occupying positions 5891 to 6889 in the PDK — 30s1202771 scaffold; and 
 the P52 microsatellite marker which, in the 5′→3′ direction, has a (ct) motif and is flanked, in 5′, by the nucleotide sequence occupying positions 9276 to 10275 in the PDK — 30s680001 scaffold (GenBank accession number: GL745189.1), and in 3′, by the nucleotide sequence occupying positions 10308 to 11307 in the PDK — 30 s680001 scaffold. 
 
     
     
         2 . The method according to  claim 1 , wherein the pair of primers specific of the microsatellite marker comprises:
 if the microsatellite marker is P80: a forward SSR primer comprising, or consisting of, between 15 and 50 consecutive nucleotides of the sequence of at most 1000 nucleotides flanking, in 5′, position 74550 in the PDK — 30 s6550963 scaffold, and a reverse SSR primer comprising, or consisting of, between 15 and 50 consecutive nucleotides of the sequence complementary to the sequence of at most 1000 nucleotides flanking, in 3′, position 74565 in the PDK — 30s6550963 scaffold; or   if the microsatellite marker is P50: a forward SSR primer comprising, or consisting of, between 15 and 50 consecutive nucleotides of the sequence of at most 1000 nucleotides flanking, in 5′, position 5871 in the PDK — 30 s1202771 scaffold (GenBank accession number: GL744456.1), and a reverse SSR primer comprising, or consisting of, between 15 and 50 consecutive nucleotides of the sequence complementary to the sequence of at most 1000 nucleotides flanking, in 3′, position 5890 in the PDK — 30 s1202771 scaffold; or   if the microsatellite marker is P52: a forward SSR primer comprising, or consisting of, between 15 and 50 consecutive nucleotides of the sequence of at most 1000 nucleotides flanking, in 5′, position 10276 in the PDK — 30 s680001 scaffold, and a reverse SSR primer comprising, or consisting of, between 20 and 35 consecutive nucleotides of the sequence complementary to the sequence of at most 1000 nucleotides flanking, in 3′, position 10307 in the PDK — 30 s680001 scaffold.   
     
     
         3 . The method according to  claim 2 , wherein the pair of primers specific for the microsatellite marker consists of:
 if the microsatellite marker is P80: a forward SSR primer comprising, or consisting of, the sequence set forth in SEQ ID NO: 4 and a reverse SSR primer comprising, or consisting of, the sequence set forth in SEQ ID NO: 5; or   if the microsatellite marker is P50: a forward SSR primer comprising, or consisting of, the sequence set forth in SEQ ID NO: 6 and a reverse SSR primer comprising, or consisting of, the sequence set forth in SEQ ID NO: 7; or   if the microsatellite marker is P52: a forward SSR primer comprising, or consisting of, the sequence set forth in SEQ ID NO: 8 and a reverse SSR primer comprising, or consisting of, the sequence set forth in SEQ ID NO: 9.   
     
     
         4 . The method according to  claim 1 , wherein analyzing the amplicons obtained in order to determine the sex of the date palm tested comprises steps of:
 separating, according to their size, the amplicons obtained by amplification of the portion of the date palm genomic DNA; and   comparing the sizes of said amplicons with the sizes of the amplicons obtained by amplification, under the same conditions, of a portion of genomic DNA of a control date palm.   
     
     
         5 . The method according to  claim 4 , wherein:
 if the amplification is carried out with a pair of primers consisting of a forward SSR primer comprising, or consisting of, the sequence set forth in SEQ ID NO: 4 and a reverse SSR primer comprising, or consisting of, the sequence set forth in SEQ ID NO: 5, the presence of an amplicon containing 194 nucleotides and/or of an amplicon containing 310 nucleotides indicates that the date palm tested is a male palm plant;   if the amplification is carried out with a pair of primers consisting of a forward SSR primer comprising, or consisting of, the sequence set forth in SEQ ID NO: 6 and a reverse SSR primer comprising, or consisting of, the sequence set forth in SEQ ID NO: 7, the presence of an amplicon containing 180, 182, 214, 223, 225 or 227 nucleotides indicates that the date palm tested is a male palm plant;   if the amplification is carried out with a pair of primers consisting of a forward SSR primer comprising, or consisting of, the sequence set forth in SEQ ID NO: 8 and a reverse SSR primer comprising, or consisting of, the sequence set forth in SEQ ID NO: 9, the presence of an amplicon containing 188, 190, 191, 193, 197 or 199 nucleotides indicates that the date palm tested is a male palm plant.   
     
     
         6 . The method according to  claim 1 , wherein analyzing the amplicons obtained in order to determine the sex of the date palm tested comprises a step of:
 sequencing the amplicons in order to determine the number of repeats of the SSR motif of each amplicon.   
     
     
         7 . The method according to  claim 6 , wherein analyzing the amplicons obtained in order to determine the sex of the date palm tested further comprises, prior to the sequencing step, a step of:
 separating, according to their size, the amplicons obtained by amplification of the portion of the date palm genomic DNA.   
     
     
         8 . The method according to  claim 1 , wherein the method is carried out on genomic DNA extracted from a sample of the date palm and wherein the date palm sample is a sample of protoplast, of callus, of embryos, of leaf, of trunk, of root, of offshoots of cutting of the date palm or any combination thereof. 
     
     
         9 . The method according to  claim 8 , wherein the date palm sample is collected from a date palm plant up to 6 to 8 years after sowing. 
     
     
         10 . A primer pair for the identification of the sex of a date palm consisting of:
 a forward SSR primer comprising, or consisting of, between 15 and 50 consecutive nucleotides of the sequence of at most 1000 nucleotides flanking, in 5′, position 74550 in the PDK — 30 s6550963 scaffold, and a reverse SSR primer comprising, or consisting of, between 15 and 50 consecutive nucleotides of the sequence complementary to the sequence of at most 1000 nucleotides flanking, in 3′, position 74565 in the PDK — 30 s6550963 scaffold; or   a forward SSR primer comprising, or consisting of, between 15 and 50 consecutive nucleotides of the sequence of at most 1000 nucleotides flanking, in 5′, position 5871 in the PDK — 30 s1202771 scaffold (GenBank accession number: GL744456.1), and a reverse SSR primer comprising, or consisting of, between 15 and 50 consecutive nucleotides of the sequence complementary to the sequence of at most 1000 nucleotides flanking, in 3′, position 5890 in the PDK — 30 s1202771 scaffold; or   a forward SSR primer comprising, or consisting of, between 15 and 50 consecutive nucleotides of the sequence of at most 1000 nucleotides flanking, in 5′, position 10276 in the PDK — 30 s680001 scaffold, and a reverse SSR primer comprising, or consisting of, between 20 and 35 consecutive nucleotides of the sequence complementary to the sequence of at most 1000 nucleotides flanking, in 3′, position 10307 in the PDK — 30 s680001 scaffold.   
     
     
         11 . The primer pair according to  claim 10  comprising:
 a forward SSR primer comprising, or consisting of, the sequence set forth in SEQ ID NO: 4 and a reverse SSR primer comprising, or consisting of, the sequence set forth in SEQ ID NO: 5; or 
 a forward SSR primer comprising, or consisting of, the sequence set forth in SEQ ID NO: 6 and a reverse SSR primer comprising, or consisting of, the sequence set forth in SEQ ID NO: 7; or 
 a forward SSR primer comprising, or consisting of, the sequence set forth in SEQ ID NO: 8 and a reverse SSR primer comprising, or consisting of, the sequence set forth in SEQ ID NO: 9. 
 
     
     
         12 . The primer pair according to  claim 10 , wherein at least one of the primers of the primer pair comprises a detectable label. 
     
     
         13 . (canceled) 
     
     
         14 . A kit for identifying the sex of a date palm comprising at least one primer pair according to  claim 10 , and instructions for identifying the sex of a date palm using a method according to  claim 1 . 
     
     
         15 . (canceled) 
     
     
         16 . A method for identifying the sex of a date palm comprising steps of:
 amplifying a portion of genomic DNA of the date palm tested using a pair of primers specific of a male-specific marker in order to obtain amplicons, and   detecting if amplicons are produced, wherein detection of amplicons indicates that the date palm tested as a male date palm,   
       wherein, in the 5′→3′ direction, the male-specific marker is located at position 74489 in scaffold PDK — 30 s6550963 and has the sequence set forth in SEQ ID NO: 18. 
     
     
         17 . The method according to  claim 16 , wherein the pair of primers specific for the male-specific marker consists of:
 a forward PCR primer comprising, or consisting of, between 15 and 18 consecutive nucleotides of the sequence of at most 2000 nucleotides flanking, in 5′, position 74489 in the PDK — 30 s6550963 scaffold, and   a reverse PCR primer comprising, or consisting of, between 15 and 18 consecutive nucleotides of the sequence complementary to the sequence of at most 2000 nucleotides flanking, in 3′, position 74506 in the PDK — 30 s6550963 scaffold.   
     
     
         18 . The method according to  claim 17 , wherein the forward PCR primer comprises, or consists of, the sequence set forth in SEQ ID NO: 19 and the reverse PCR primer comprises, or consists of, the sequence set forth in SEQ ID NO: 20. 
     
     
         19 . The method according to  claim 16 , wherein the method is carried out on genomic DNA extracted from a sample of the date palm and wherein the date palm sample is a sample of protoplast, of callus, of embryos, of leaf, of trunk, of root, of offshoots of cutting of the date palm or any combination thereof. 
     
     
         20 . The method according to  claim 19 , wherein the date palm sample is collected from a date palm plant up to 6 to 8 years after sowing. 
     
     
         21 . A primer pair for the identification of the sex of a date palm consisting of:
 a forward PCR primer comprising, or consisting of, between 15 and 18 consecutive nucleotides of the sequence of at most 2000 nucleotides flanking, in 5′, position 74489 in the PDK — 30 s6550963 scaffold, and   a reverse PCR primer comprising, or consisting of, between 15 and 18 consecutive nucleotides of the sequence complementary to the sequence of at most 2000 nucleotides flanking, in 3′, position 74506 in the PDK — 30 s6550963 scaffold.   
     
     
         22 . The primer pair according to  claim 21 , wherein the forward PCR primer comprises, or consists of, the sequence set forth in SEQ ID NO: 19 and the reverse PCR primer comprises, or consists of, the sequence set forth in SEQ ID NO: 20. 
     
     
         23 . The primer pair according to  claim 21  or  claim 22 , wherein at least one of the primers of the primer pair comprises a detectable label. 
     
     
         24 . (canceled) 
     
     
         25 . A kit for identifying the sex of a date palm comprising at least one primer pair according to  claim 21  and instructions to identify the sex of a date palm using a method according to  claim 16 . 
     
     
         26 . (canceled) 
     
     
         27 . (canceled)